TGF-β belongs to a large super-family of multifunctional polypeptide factors. The TGF-β family includes three genes, TGF-β1, TGF-β2 and TGF-β3, which are pleiotropic modulators of cell growth and differentiation, embryonic and bone development, extracellular matrix formation, hematopoiesis, immune and inflammatory responses. For example, TGF-β1 inhibits the growth of many cell types, including epithelial cells, but stimulates the proliferation of various types of mesenchymal cells.
The TGF-β genes have high homology with one another. In mammals, the TGF-β super-family includes various TGF-β genes, as well as the embryonic morphogenes, such as the family of the activins, inhibins, “Mullerian Inhibiting Substance”, and bone morphogenic protein (BMP). See Roberts and Spom, “The Transforming Growth Factor-βs in Peptide Growth Factors and Their Receptors. I.” Handbook of Experimental Pharmacology, vol. 95/I, Springer-Verlag, Berlin, 1990, pp 419-472. Each member of the TGF-β family exerts a wide range of biological effects on a large variety of cell types, e.g., they regulate cell growth, morphogenesis, differentiation, matrix production and apoptosis. Lagna et al., Nature, 1996, 383, 832-836. TGF-β acts as a growth inhibitor for many cell types and is believed to play a central role in the regulation of embryonic development, tissue regeneration, immuno-regulation, as well as in fibrosis and carcinogenesis.
In addition, TGF-β induces the synthesis of extracellular matrix (ECM) proteins, modulates the expression of matrix proteinases and proteinase inhibitors and changes the expression of integrins. ECM is a dynamic superstructure of self aggregating macromolecules including fibronectin, collagen and proteoglycan. It is believed that ECM is the chief pathologic feature of fibrotic diseases. ECM disorder has also been proposed to play a central role in pathogenesis disorders such as hypertensive vascular disease and diabetic renal disease. Sato et al., Am. J. Hypertens., 1995, 8, 160-166 (1995); Schulick et al., Proc. Natl. Acad. Sci., 1988, 95, 6983-6988. Moreover, TGF-β is expressed in large amounts in many tumors. Derynck, Trends Biochem. Sci., 1994, 19, 548-553. This strong occurrence in neoplastic tissues could indicate that TGF-β is strategic growth/morphogenesis factor which influences the malignant properties associated with the various stages of the metastatic cascade. TGF-β inhibits the growth of normal epithelial and relatively differentiated carcinoma cells, whereas undifferentiated tumor cells which lack many epithelial properties are generally resistant to growth inhibition by TGF-β. Hoosein et al., Exp. Cell. Res., 1989, 181, 442-453; Murthy et al., Int'l. J Cancer, 1989, 44, 110-115. Furthermore, TGF-β1 is believed to potentiate the invasive and metastatic potential of a breast adenoma cell line (Welch et al., Proc. Natl. Acad. Sci., 1990, 87, 7678-7682), which indicates a role of TGF-β1 in tumor progression.
The cellular effects of TGF-β are exerted by ligand-induced hetero-oligomerization of two distantly related type I and type II serine/threonine kinase receptors, TGF-βR-I and TGF-βR-II, respectively. Lin et al., Trends Cell Biol., 1993, 11, 972-978; Massague et al., Cancer Surv., 1996, 27, 41-64; Dijke et al., Curr. Opin. Cell Biol., 1996, 8, 139-145. The two receptors, both of which are required for signaling, act in sequence: TGF βR-I is a substrate for the constitutively active TGF-βR-II kinase. Wrana et al., Nature, 1994, 370, 341-347; Wieser et al., EMBO J., 1995, 14, 2199-2208. Upon TGF-β1 binding, the type II receptor phosphorylates threonine residues in GS domain of ligand occupied type 1 receptor or activin like kinase (ALK5), which results in activation of type I receptors. The TGF-β1 type I receptor in turn phosphorylates Smad2 and Smad3 proteins which translocate to the nucleus and mediate intracellular signaling. The inhibition of ALK5 phosphorylation of Smad3 reduces TGF-β1 induced extracellular matrix production. Krettzchmar et al., Genes Dev., 1997, 11, 984-995; Wu et al., Mol. Cell. Biol., 1997, 17,2521-2528.
TGF-β is also a powerful and essential immune regulator in the vascular system capable of modulating inflammatory events in both leuko and vascular endothelial cells. Shull et al., Nature, 1992, 359, 693-699. It is also involved in the pathogenesis of chronic vascular diseases such as atherosclerosis and hypertension. Grainger & Metcalfe et al., Bio. Rev. Cambridge Phil. Soc., 1995, 70, 571-596; Metcalfe et al., J. Human Hypertens., 1995, 9, 679.
Genetic studies of TGF-β-like signaling pathways in Drosophila and Caenorhabditis elegans have led to the identification of mothers against dpp (Mad) and sma genes, respectively. Sekelsky et al., Genetics, 1995, 139, 1347-1358; Savage et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 790-794. The products of these related genes perform essential functions downstream of TGF-β like ligands acting via serine/threonine kinase receptors in these organisms. Wiersdorf et al., Development, 1996, 122, 2153-2163; Newfeld et al., Development, 1996, 122, 2099-2108; Hoodless et al., Cell, 1996, 85, 489-500.
Vertebrate homologs of Mad and sma have been termed Smads or MADR genes. Derynck et al., Cell, 1996, 87, 173; Wrana et al., Trends Genet., 1996, 12, 493-496. Smad proteins have been identified as signaling mediators of TGF-β super family. Hahn et al., Science, 1996, 271, 350-353. Genetic alterations in Smad2 and Smad4/DPC4 have been found in specific tumor subsets, and thus Smads may function as tumor suppressor genes. Hahn et al., Science, 1996, 271, 350-353; Riggins et al., Nature Genet., 1996, 13, 347-349; Eppert et al., Cell, 1996, 86, 543-552. Smad proteins share two regions of high similarity, termed MH1 and MH2 domains, connected with a variable proline-rich sequence. Massague, Cell, 1996, 85, 947-950; Derynck et al., Curr. Biol., 1996, 6, 1226-1229. The C-terminal part of Smad2, when fused to a heterologous DNA-binding domain, was found to have transcriptional activity. Liu et al., Nature, 1996, 381, 620-623; Meersseman et al., Mech. Dev., 1997, 61, 127-140. The intact Smad2 protein when fused to a DNA-binding domain, was latent, but transcriptional activity was unmasked after stimulation with ligand. Liu et al., supra.
TGF-β initiates an intracellular signaling pathway leading ultimately to the expression of genes that regulate the cell cycle, control proliferative responses, or relate to extracellular matrix proteins that mediate outside-in cell signaling, cell adhesion, migration and intercellular communication.
TGF-β is also an important mediator of diabetic nephropathy, a common complication in patients with either type 1 or type 2 diabetes mellitus. Ziyadeh et al., Proc. Natl. Acad. Sci., 2000, 97, 8015-8020 evaluated the role of renal TGF-β in the development of chronic structural and functional changes of diabetic nephropathy by assessing the response of db/db mice to chronic treatment with neutralizing anti-TGF-β1 and generalized (tubular and glomerular) up-regulation of TGF-β type II receptor. The antibody effectively prevented increases in renal expression of matrix genes including type IV collagen and fibronectin and may have also stimulated matrix degradative pathways because TGF-β suppresses the activity of metalloproteinases and increase the expression of protease inhibitors such as plasminogen activator inhibitor-1 (PAI-1).
There exists a need for effective therapeutic agents for inhibiting TGF-β activity, as well as for inhibiting the phosphorylation of smad2 or smad3 by TGF-β type I or activin like kinase (ALK5) receptor and for preventing and treating disease states mediated by the TGF-β signaling pathway in mammals. In particular, there continues to be a need for compounds which selectively inhibit TGF-β.